Electrophoresis of nucleic acid fragments

ABSTRACT

A method for electrophoresis of nucleic acid fragments present in the solution which contains an amount, e.g., 0.2% or more, of a reagent, e.g., glycerol, dithiolthreitol (DTT) and trehalose or other sugars, which interact to form a complex with borate or boric acid. The method includes applying the solution to an electrophoretic gel and electrophoresing those fragments into the gel in the presence of a buffer lacking boric acid, or a derivative thereof, which forms a chelate complex with the reagent and thereby causes distortion of electrophoresis of the fragments in a gel including such a buffer.

This is a division of application Ser. No. 07/928,852 filed Aug. 10,1992 hereby incorporated by reference in its totality (includingdrawings), which application is a continuation-in-part of Fuller, U.S.Ser. No. 07/862,734, filed Apr. 3, 1992, now U.S. Pat. No. 5,314,595,hereby incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to methods and kits for electrophoresis ofnucleic acid fragments, particularly those methods and kits useful forelectrophoresing nucleic acid fragments contained in a solution whichincludes glycerol.

BACKGROUND OF THE INVENTION

Fuller, 16 Comments 1989 reports that the presence of glycerol in asample loaded on an electrophoretic gel may cause artifacts, such asdistortion of nucleic acid fragments in DNA sequencing gels. Thedistortion is said to resemble a bulge in the sequencing gel, andrenders the 400-600 nucleotide region of the gel unreadable. Fullerstates that because of the distortion caused by the presence ofglycerol, United States Biochemical Corporation supplies a sequencingenzyme, SEQUENASE Version 2.0 T7 DNA polymerase, at a high enoughconcentration so that no glycerol distortion occurs when the enzyme isdiluted prior to use.

Carninci et al. 18 Nucleic Acids Research 204, 1989 describe a standardsequencing gel system using Tris/Borate/EDTA buffer (TBE). It alsodescribes a discontinuous buffer system using Tris-sulphate andTris-borate. The Tris-sulphate is used as a running gel buffer, andTris-borate as a tank buffer.

Richards, et al., 12 Analytical Biochemistry 452, 1965 and Peacock andDingman, 6 Biochemistry 1818, 1967 describe electrophoresis ofribonucleic acid in polyacrylamide gels and resolution of multiple RNAspecies by polyacrylamide gel electrophoresis. Richards, et al.,describe use of Tris-HCl buffer as well as acetic, cacodylic, diethylbarbituric, and glycyl glycine buffers. Peacock and Dingman describe useof Tris-EDTA and boric acid buffers for electrophoresis. The RNA speciesare not provided in glycerol-containing samples.

Ansorge and Barker, 9 J. Bioc. Biop. Meth. 33, 1984 describe use ofTris/Tricine buffer and an ammediol system for electrophoresis of Maxamand Gilbert DNA sequencing products. Such products are provided insamples without glycerol.

Brumley and Smith, 19 Nuc. Acid. Res. 4121, 1991 describe use of aborate buffer for a sequencing gel.

SUMMARY OF THE INVENTION

The present invention concerns use of an electrophoretic buffer, forelectrophoresis of nucleic acids, which does not form a chelate complexwith glycerol or other reagents. Such complexes, e.g., between boricacid and glycerol, are described by Cotton and Wilkinson, AdvancedInorganic Chemistry, 1980, John Wiley & Sons, p. 298. Other suchcomplexes may be formed between borate and various sugars, such asethylene glycol, trehalose or dithiothreitol (DTT) which may be used asstabilizing agents for an enzyme (e.g., trehalose is used by Quadrantfor stabilizing a dried enzyme preparation). The presence of such acomplex within an electrophoretic gel causes distortion of negativelycharged DNA molecules within the gel. The complex is negatively chargedunder electrophoretic gel conditions and migrates through the gel alongwith the negatively charged DNA. It is present in sufficient amount tooverload the gel, causing distortion of adjacent DNA bands within thegel.

Applicant has discovered that substitution of a different weak acid forboric acid in an electrophoretic gel buffer eliminates gel distortion.The invention features a method for electrophoresis of nucleic acidfragments, such as those produced during DNA sequencing procedures, byuse of a buffer which does not form a complex with glycerol or otherreagents. This enables use of glycerol or the other reagents at highconcentrations in DNA sequencing reactions or other reactions concerningnucleic acids. Such high concentrations are advantageous because theyare more convenient and they increase the stability of the enzymeswithin a reaction mixture.

Thus, in the first aspect the invention features a method forelectrophoresis of nucleic acid fragments present in the solution whichcontains an amount, e.g., 0.2% or more, of a reagent, e.g., glycerol,DTT, and trehalose or other sugars, which interacts to form a complexwith borate or boric acid. Generally, boric acid may react with any1,2-diol compound, and the stability of the product depends on therelative orientation of the alcohols. Thus, complexes with trans glycolsare weaker than with cis glycols. Examples of such 1,2-diols (glycols)include ethylene glycol, propylene glycol, butylene glycol, threitol,erythritol, dithioerythritol, pinacol, ribose, mannitol, glucitol,ribitol, sorbitol, inositols, SPAN® and TWEEN® detergents, sorbose,ascorbic acid, FICOLL®, dextran, and derivatives thereof. Various sugarswill also react, including glyceraldehyde, erythrose, threose,arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose,idose, galactose, talose, fructose, ribulose, xylulose, fucose,rhamnose, fructose, glycosides of these, sucrose and otheroligosaccharides, polysaccharides, cellobiose, maltose, lactose,trehalose, gentiobiose, melibiose, cellulose, and starches.Amino-sugars, acylaminosugars and glycosides may also react. That is,any compound having a 1,2-diol moiety which reacts with borate to forman anionic chelate complex is best avoided in gel compositions andreagents run into a gel. The method includes applying the solution to anelectrophoretic gel and electrophoresing those fragments into the gel inthe presence of a buffer lacking boric acid, or a derivative thereof,which forms a chelate complex with the reagent and thereby causesdistortion (i.e., makes reading of the DNA sequence more difficult thanif there were no distortion) of electrophoresis of the fragments.

In preferred embodiments, the fragments are produced during an enzymaticDNA sequencing reaction; the gel is a DNA sequencing gel; the reactionis performed in the presence of the glycerol; and the acid portion ofthe buffer is selected from acetic acid, carbonic acid, glycine, serine,taurine, tricine, and bicine. The basic portion may be Tris or ammediolor any other equivalent buffer components.

In a related aspect, the invention features a kit for DNA sequencingwhich includes reagents, e.g., DNA polymerase, necessary for DNAsequencing, and an electrophoretic buffer selected from those describedabove. Most preferably, the polymerase is provided at a concentration ina glycerol-containing buffer which does not need dilution prior to use(e.g., within a microtitre well format). By not needing dilution ismeant that the DNA polymerase can be used directly in a sequencingreaction without a step of dilution of the enzyme prior to addition toother components and in a volume that can be readily and accuratelytransferred by commonly available pipetting devices (2 μl of 50%glycerol solution).

Prior to this invention, SEQUENASE DNA polymerase and Delta Taq DNApolymerase were provided at high concentration (e.g., at 13 U/μl forSEQUENASE DNA polymerase and 32 U/μl for Delta Taq DNA polymerase) in asequencing kit (in 50% glycerol) and the customer was forced to diluteit 8-fold (e.g., to 1.6 U/μl or 4 U/μl for SEQUENASE DNA polymerase andDelta Taq DNA polymerase, respectively) prior to use. This represents acompromise among convenience, stability and gel readability. Whendiluted in the labeling reaction mixture (without glycerol), SEQUENASEDNA polymerase remains stable only 10-15 minutes at room temperature inthe first step of the sequencing reaction, but the reaction can becompleted in 5 minutes and the gel distortion is acceptably small. Thepolymerase, however, is stable if diluted in the presence of glycerol.

The present invention eliminates the distortions on the gels, by using anew gel buffer in place of the traditional TBE buffer. The use of thisnew buffer provides the following advantages (other are exemplifiedbelow):

1. The SEQUENASE or Delta Taq DNA polymerase can be supplied pre-dilutedin 50% glycerol, eliminating a tedious step in the sequencing protocol.It can simply be added directly from the kit vial in a convenient volumeof 2 μl.

2. Enzyme dilution buffer is not required in a sequencing kit.

3. The enzyme can be much more stable in use, allowing more leeway (intime and temperature) in the manner the reactions are run. This allowseasier dispensing of enzyme by slow automated equipment during use.There are distinct improvements in sequencing results with higherconcentrations of glycerol, especially with double-stranded templates.

4. Contaminants in the template which may destabilize the enzyme areless troublesome.

5. The end user of a typical 100 test DNA sequencing kit will preferreceiving a vial containing 200 μl of enzyme over the current 25 μl.This way, it is possible to run just one reaction without wasting enzymein dilution.

6. Elimination of the gel distortion caused by glycerol is particularlyuseful for procedures which make use of added restriction enzymes andgene 6 exonuclease, such as lambda DNA sequencing.

7. A microtiter-plate format sequencing kit can be more readilyproduced. The reagents (including enzyme) can be pre-dispensed in96-well plates and used to sequence 1-12 templates simultaneously.Unused portions of the plate can be returned to the freezer for lateruse.

8. When sufficient glycerol is added to reactions run with SEQUENASE T7DNA Polymerase, the termination reactions can be run at high temperatureby pre-warming the termination reaction vials to 70° C. This hightemperature may eliminate template secondary structure problems.

It is these advantages that make possible other aspects of the inventionin which a thermolabile DNA polymerase (i.e., one which loses activityabove about 37° C., e.g., T7 DNA polymerase, and Klenow) can be used inthe labelling and/or termination steps of a DNA sequencing reaction athigh temperatures of 37° C. and 60° C. respectively. Such sequencingprocedures provide superior results to those previously obtained, asdiscussed below.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

The drawings will first briefly be described.

DRAWINGS

FIGS. 1A and 1B are a reproduction of an autoradiogram of a DNAsequencing gel formed in the presence of a borate buffer showing thatglycerol causes gel distortions;

FIGS. 2A and 2B are a reproduction of an autoradiogram of a DNAsequencing gel formed in the absence of a borate buffer showing thatglycerol causes no distortion on a gel made using a glycerol-tolerantbuffer;

FIGS. 3A and B are reproductions of autoradiograms of a DNA sequencinggel in which DNA sequencing reactions are run in the absence or presenceof glycerol; and

FIG. 4 is a graphical representation showing that glycerol stabilizesthe activity of a T7 DNA polymerase;

FIG. 5 is a diagrammatic representation of multiple binding sites on aDNA to be sequenced;

FIG. 6 is a reproduction of an autoradiogram of a DNA sequencing gelshowing the advantage of use of a high temperature for a labellingreaction in a DNA sequencing procedure.

FIG. 7 is a reproduction of an autoradiogram of a DNA sequencing gelshowing the ability to use high temperatures for termination reactionsin a DNA sequencing procedure.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Gel Electrophoresis

Current methods for sequencing DNA rely on electrophoresis gels toresolve DNA fragments according to their size or molecular weight, Maxamand Gilbert, 65 Methods in Enzymology 499, 1980, Maxam and Gilbert, 74Proc. Nat. Acad. Sci. USA 560, 1977, Sanger, et al., 74 Proc. Nat. Acad.Sci. USA 5463, 1977. A population of DNA fragments is prepared in such away that all the fragments have a particular sequence at one end, and atthe other end have generally only one (of the four possible)nucleotides. Thus, the presence of a fragment containing 50 nucleotides(in addition to a region known as a primer) in the population ofadenosine-terminated fragments indicates that adenosine is present inthe 50th position of the sequence.

High-resolution separation of DNA fragments by size is an essentialcomponent of a sequencing method. Sequencing gels are typically largerand thinner than other kinds of electrophoresis gel, so that highpossible resolution is achieved, resulting in the determination of largenumbers of bases in a single electrophoresis run. Gels typically resolve150-300 nucleotides in a single run, but there have been reports ofresolving 500 or more bases using particularly long gels, or specialapparatus. Tabor and Richardson, 84 Proc. Natl. Acad. Sci. USA 4767,1987, Sieminiak et al., 192 Anal. Biochem. 441, 1991.

Examples of sequencing gels now in use generally include 6-8%polyacrylamide crosslinked with N, N'-methylene bisacrylamide (20:1acrylamide to bisacrylamide to bisacrylamide by weight), 7-8.3M urea andTBE buffer (10.8 g tris (hydroxymethyl) aminomethane ("Tris"), 0.93 gdisodium EDTA, and 5.5 g boric acid per liter of solution) to give finalconcentrations of 0.09M Tris, 0.09M boric acid and 0.0025M EDTA. Thisbuffer was first described by Peacock and Dingman in 6 Biochemistry1818, 1967 for the separation of RNA species by polyacrylamide gelelectrophoresis. The addition of urea to this buffer to denature the DNAwas described by Maniatis, et al. in 14 Biochemistry 3787, 1975, and forsequencing DNA by Air, et al. in 108 J. Mol. Biol. 519, 1976.

The composition of sequencing gels has not changed significantly since1976, although some workers use formamide, alternative crosslinkers, andreagents which bind the gel to glass.

The DNA Polymerase used for performing a sequencing reaction to form thedesired DNA fragment preparations is stored in 50% glycerol to keep itstable at reaction temperatures or during storage at -20° C. It isdifficult to accurately transfer volumes of less than 2 μl of 50%glycerol. Thus, it is necessary to dilute the DNA polymerase immediatelyprior to use, thereby diluting the glycerol to acceptable levels.Alternatively, the samples are purified by precipitation with ethanolprior to loading on the gel.

While these measures eliminate the gel-distortion problem, they aresomewhat cumbersome, and time consuming. Thus, the present inventionfeatures a new gel formulation which tolerates glycerol in the sample.Since the polymerase can be maintained in high concentrations ofglycerol at all times, potential problems which result from enzymeinactivation are eliminated.

When glycerol is applied to an ordinary TBE-buffered DNA sequencing gel,it complexes with boric acid and migrates through the gel. If thequantity of glycerol is large enough to overload the gel, it distortsthe region of the gel in which it migrates. The degree of distortiondepends on the amount of glycerol loaded and the size of the gel. Whenfollowing the nominal protocol used for the SEQUENASE DNA sequencingkits, 10.7 μg of glycerol is loaded into each sequencing gel lane alongwith about 0.25 μg of DNA. This amount of glycerol does not normallyinterfere with DNA migration on the gel. If the enzyme is diluted in abuffer containing 50% glycerol, more than 85 μg of glycerol is presentin the sample applied to the lane. This quantity is enough to severelydistort the pattern of DNA band migration. Typical amounts of glycerolloaded on a sequencing gel are listed in Table 1.

                  TABLE 1                                                         ______________________________________                                                                     Sequenase Kit,                                                                         96-well                                 Glycerol   Sequenase                                                                              Taquence no Enzyme                                                                              Kit as                                  Concentration (%)                                                                        Kits     Kits     Dilution described                               ______________________________________                                        Enzyme     50       50       50       50                                      Diluted Enzyme                                                                           6.25     6.25     50       50                                      Labeling Reaction                                                                        0.806    0.714    6.45     25.9                                    Termination                                                                              0.470    0.357    3.76     15.9                                    Reactions                                                                     Final Sample                                                                             0.282    0.238    2.26     9.85                                    Amount of Glycerol Loaded on Typical Electrophoresis Gel Lane                 (3 μl)                                                                     μl Glycerol per lane                                                                  0.008    0.007    0.068    0.295                                   μg Glycerol per lane                                                                  10.7     9.00     85.3     372                                     ______________________________________                                    

Appropriate gel buffers can be determined by routine experimentation tofind those which are useful in DNA separations and yet do not complexwith glycerol or various sugars to form a distorting complex. Forexample, for the acid component weak acids can be used, including thefollowing acids, which serve to replace boric acid in DNA sequencinggels: acetic acid, carbonic acid, glycine, serine, taurine, tricine(N-tris (hydroxyethyl) methylglycine), and bicine (N,N-bis(2-hydroxyethyl)glycine). Of these, the stronger acids (acetic andcarbonic) yield buffers with higher conductivity than desired forroutine use. The weakest acid, glycine, yields a buffer with a pHsomewhat higher than normally used, although results are acceptable. Theothers all yield buffers with electrophoretic properties essentiallyidentical to those of borate-containing buffers, but the addition ofhigh concentrations of glycerol has no effect on the gel resolution.Glycine and taurine are preferred at present since they are lessexpensive than some of the other acids, and more soluble thanD,L-serine. Other organic and inorganic acids could also be used,particularly those with a pKa value between 7 and 10 at 40°-50° C. Forexample, TAPS (3- N-tris-(hydroxymethyl) methyl-amino!-propanesulfonicacid), CHES (2-(N-cyclohexylamino) ethanesulfonic acid), AMPSO (3-dimethyl (hydroxymethyl)-methylamino!-2-hydroxypropanesulfonic acid),CAPSO (3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid), CAPS(3-N-(Cyclohexylamino)-propanesulfonic acid), Glycylglycine, Threonine,allo-Threonine, arginine, 2-aminoethyl sulfuric acid, and glutamic acid.

EXAMPLES

The following buffers have been prepared and tested for running DNAsequencing gels. Typically, they were used as follows:

1. Buffer was prepared in concentrated form (usually 10-foldconcentrate) with the quantities of reagents shown in Table 2.

2. A sequencing gel was prepared by mixing 7.6 g acrylamide, 0.4 g N,N'-methylene bisacrylamide, 42 g urea, concentrated buffer and water toa final dissolved volume of 100 ml. The volume of concentrated bufferwas usually 10 ml but varied in actual experiments. Actual volumes usedare listed in Table 2. The mixture was dissolved by gentle stirring atroom temperature (about 20° C.).

3. The mixture was filtered either through Whatman #1 filter paper or a0.4 μm nitrocellulose filter. Gel molding plates, spacers and comb wereassembled using tape and clamps by standard procedures. The gelapparatus used is a model STS 45 from IBI division of Kodak. Gels wereapproximately 42 cm high, 37 cm wide and 0.4 mm thick, run with a64-well "sharkstooth" comb.

4. When ready, 1.0 ml of 10% ammonium persulfate and 25 μl ofN,N,N',N'-tetramethylethylenediamine were added to the gel formingsolution and the gel poured immediately. Gels were typically used within2-3 hours of pouring but sometimes used after sitting overnight at roomtemperature.

5. Gels were loaded with sequencing reaction products, labeled with ³⁵ Sprepared using the USB SEQUENASE® Version 2.0 DNA sequencing kit andM13mp18 DNA for template.

6. Running buffer (1000 ml) was prepared by diluting the sameconcentrated buffer by the same ratio as for preparing the gel. Thus,the same buffer was present in the upper and lower chambers and in thegel itself. Gels were run at constant power (60-70 watts) until thebromophenol blue dye reached the bottom, typically 2-3 hours. Thevoltage and current required to maintain 60-70 watts varied inaccordance with the conductivity of the buffer. Gels prepared withbuffers of high conductivity generally ran slower.

7. Gels and sequencing reactions were also prepared for running on theApplied Biosystems Model 373A automated DNA sequencing instrumentaccording to the instructions of the instrument manufacturer except forthe substitution of buffer. These gels also were free ofglycerol-induced artifacts and gave normal sequencing results.

8. These buffers also work for running horizontal agarose gels for theseparation of DNAs much like the currently popular TBE and TAE buffers.

                                      TABLE 2                                     __________________________________________________________________________                 Composition; g/500 ml                                                                       Conductivity                                                                        Dilution                                                  Buffer Concentrate                                                                          (of a 1:10                                                                          for Gel                                          Weak acid used                                                                         Weak  Na.sub.2                                                                          pH  dilution)                                                                           ml/100 ml                                    Buffer                                                                            (pKa at 45° C.)                                                                 Acid                                                                             Tris                                                                             EDTA                                                                              (1:10)                                                                            μmho/cm                                                                          of Gel                                       __________________________________________________________________________    1 (TBE)                                                                           Boric Acid (9.1)                                                                       28 54 4.5 8.16                                                                              755   10                                           2   Bicine (7.9)                                                                           50 54 4.5 8.20                                                                              1000  6.67                                         3   D,L-Serine (8.7)                                                                       35 54 4.5 8.47                                                                              734   10                                           4   Acetic Acid (4.8)                                                                      .sup.a                                                                           54 4.5 8.10                                                                              1150  10                                           5   Carbonic Acid (6.3)                                                                    .sup.b                                                                           54 4.5 8.23                                                                              1070  10                                           6   Glycine (9.3)                                                                          50 54 4.5 8.53                                                                              700   10                                           7   Glycine (9.3)                                                                          33.75                                                                            20 4.5 8.45                                                                              435   15                                           8   Glycine (9.3)                                                                          80 108                                                                              9   8.6 1500  5                                            9   Tricine (7.6)                                                                          15 54 4.5 8.23                                                                              1075  6.67                                         10  Taurine (8.6)                                                                          10 54 4.5 8.85                                                                              560   10                                           11  Taurine (8.6)                                                                          18 54 4.5 8.65                                                                              750   10                                           12  Taurine (8.6)                                                                          20 54 4.5 8.70                                                                              800   10                                           13  Taurine (8.6)                                                                          25 54 4.5 8.60                                                                              890   10                                           14  Taurine (8.6)                                                                          35 54 4.5 8.45                                                                              950   10                                           __________________________________________________________________________     .sup.a Acetic acid was added until the pH of the concentrated buffer was      8.3. This required approximately 12 ml of acetic acid.                        .sup.b The solution of Tris and EDTA was bubbled with CO.sub.2 until the      pH of the concentrated buffer was 8.3.                                   

Example 1

Referring to FIG. 1, the gel picture demonstrates that glycerol causessequencing gel distortions. One set of four DNA sequencing reactions wasrun using the SEQUENASE® Version 2.0 DNA sequencing kit (USB) usingtemplate DNA and α-³⁵ S dATP following the methods supplied with thekit. The final reaction products (G, A, T and C reactions, 10 μl each)were divided into two equal portions and 1 μl of 50% glycerol was addedto one portion, resulting in a total glycerol concentration of about8.6%. A 3 μl portion of these were loaded onto an 8% polyacrylamidesequencing gel prepared with normal TBE buffer. The four left-hand lanescontained the added glycerol such that 0.26 μl or 324 μg of glycerol wasloaded into each lane. The distortion in these lanes is so severe thatit also distorted the four adjacent lanes which carried the sampleswithout added glycerol (total glycerol concentration about 0.28% so thata total of 0.0085 μl or 10.7 μg glycerol was loaded onto each lane).Other experiments have shown that while samples containing this amountof glycerol are essentially free of distortion, ones with twice thisamount and above exhibit distortions which can interfere withreadability of the gel.

Example 2

Referring to FIG. 2, the gel picture demonstrates that glycerol causesno distortion on a gel made using a glycerol-tolerant buffer. Sampleswere prepared identically with those discussed above in Example 2, andwere run on an 8% polyacrylamide gel using a buffer which containedtaurine in place of boric acid. This buffer has the composition andproperties of buffer 11 in Table 2. There is no evidence of anydistortion even with this very high concentration of glycerol.

Example 3

Referring to FIGS. 3A and 3B, the gel pictures demonstrate that DNAsequencing results can be improved if the reactions are run with addedglycerol. Sequencing reactions were run as described in Example 1 exceptthat the labeling step was run at 41° C. for 10, 20 or 40 minutes asindicated. In FIG. 3A, the enzyme was diluted normally so that theconcentration of glycerol present during the labelling step was 0.8%. InFIG. 3B the enzyme was diluted using dilution buffer that contained 50%glycerol so that the concentration of glycerol present during thelabelling step was 6.25%. The gel was identical to the one described inFIG. 2. Under these conditions, sequencing reactions run poorly with lowglycerol concentration but the performance was restored when sufficientglycerol was added. If these sequences had been run on a normal TBE gel,severe distortions would have been evident.

Example 4

Referring to FIG. 4, the graph demonstrates that glycerol stabilizes theactivity of a modified T7 DNA polymerase. Samples of SEQUENASE Version2.0 T7 DNA polymerase at a concentration of 0.5 Units/μl were incubatedat 37° C. for the times indicated in 10 mM Tris-HCl (pH 7.5), 5 mM DTTand 0.5 mg/ml bovine serum albumin containing either no added glycerol,15% glycerol or 50% glycerol as indicated. After incubation at 37° C.,the samples were chilled on ice and assayed by a standard procedure.Assay reaction mixtures (100 μl) contained 40 mM Tris-HCl pH 7.5, 10 mMMgCl₂, 5 mM DTT, 0.3 mM dGTP, dCTP, dATP and ³ H!dTTP, 5 μg of M13mp18single-stranded DNA pre-annealed to 5 pmol universal (-20) primer andenzyme. The enzyme (10 μl) was added last to the pre-warmed (37° C.)reaction mixture and incubation was for 1 minute at 37° C. The reactionwas stopped by the addition of 20 μl of 5 mg/ml fish-sperm DNA and 3 mlof 1N HCl, 0.1M sodium pyrophosphate and acid-insoluble radioactivitydetermined by filtration using glass-fiber filters. Enzyme incubated inthe absence of glycerol was inactivated within 5 minutes under theseconditions but remained at least 50% active even after 40 minutes in thepresence of 50% glycerol.

Example 5 Buffers without EDTA

Buffers without EDTA present in them are particularly useful in thisinvention, e.g., buffers containing 40 g glycine, 54 g Tris in 500 ml,pH 8.6 and having a conductivity of 500 μmho/cm, can be used at a 1:10dilution. Without EDTA bands at the bottom of gels are improved inseparation and clarity.

Example 6 Increasing Labeling Reaction Stringency

Another advantage of the ability to use glycerol to store a DNApolymerase prior to use in sequencing capitalizes on the increasedthermal stability of some DNA polymerases in the presence of highconcentrations of glycerol. For example, genetically modified T7 DNApolymerase (Sequenase Version 2.0 DNA polymerase) is much more stable at37° C. in the presence of 15-50% glycerol. Since this enzyme is used attemperatures of 37° C. for sequencing (or higher, see below), improvedstability at this temperature may improve sequencing results, as well asmake sequencing more convenient.

That is, there is a benefit from the ability to add glycerol to DNAsequencing reactions in that the reactions can be run for longer periodsof time and at warmer temperatures with less concern for polymerasestability. The original two-step sequencing protocol for sequencing withmodified T7 DNA polymerase called for incubating the first (labeling)step for up to 5 minutes at about 20° C. (room temperature) or cooler.Incubations much warmer than this have previously resulted in severesequencing problems. If the labeling step is performed at 41° C. aftereven brief incubations, a distinct pattern of artifact bands appears,which is probably the result of enzyme inactivation. However, if thepolymerase is pre-diluted in a 50% glycerol-containing storage buffer,the sequences obtained with a 41° C. labeling step are good, even withlabeling reaction times as long as 40 minutes. This allows moreconvenient sequencing protocols since the reactions can be run withoutstrict attention to either incubation time or temperature.

More important is the effect temperature can have on the overallperformance of sequencing reactions. Chain-termination sequencingrequires the use of a short primer (usually a synthetic oligonucleotide)to initiate synthesis of DNA. While efforts may be taken to ensure thathis oligonucleotide primes synthesis at a unique site in the templateDNA, it is never entirely feasible to eliminate the possibility ofmultiple priming sites when determining new sequence. As shown in FIG.5, even a few nucleotides, if at the 3' end of the primer, mighteffectively prime synthesis at an unintended site.

When using a two-step protocol, the primer itself is directly extendedduring the labeling step. It is in this step that primer specificity orlack of specificity is important. During this step, the primer isextended until the supply of nucleotide is exhausted, typically anaverage of 20-50 nucleotides. If this step is run under low temperatureconditions (low stringency), the priming may occur not only at theintended, 100% matching site, but also at sites where fewer bases matchat the 3' terminus of the primer, perhaps as few as 5-7 nucleotides.Priming at these sites will be exhibited as a second sequence along withthe sequence from the 100% matched site. This secondary sequence may befainter than the primary one, especially if a large excess of primer isavoided, but it may well make reading the primary sequence difficult orimpossible. If the temperature (stringency) of the labeling reaction canbe increased, priming at the secondary site may be eliminated, makingthe particular primer/template combination usable.

An example of low-stringency priming of a mismatched primer is shown inFIG. 6. In one experiment, when the labeling step was run at lowtemperature, a double sequence is obtained, one from the primary(matched) site and one from the secondary (10-base) site. When thetemperature of the labeling reaction was raised to 47° C., uniquesequence was obtained, indicating that the primer only anneals at itsprimary site at this elevated temperature.

Specifically, a series of identical sequencing reactions (with Mn²⁺)were run, varying only the temperature of the labeling reaction. Thetemplate was M13mp18 single-stranded DNA and the primer was an 18-merwith the sequence 5'-TTTTCATCGGCATTTTCG (SEQ ID NO:1). It matches thesequence of single-stranded M13mp18 at position 2464-2447 and the10-nucleotide sequence at its 3' end also matches the same template atposition 3703-3694. Thus, double-priming of the type depicted in FIG. 5may occur with this primer when sequencing (labeling) reactions are runat low stringency, but may not occur when stringency is high enough.Labeling reactions were run using a 5:1 primer: template mole ratio withpre-warming of the reaction mixture at the indicated temperature priorto adding polymerase. Incubation was continued for 5 minutes andtermination reactions were run at 37° C. for 5 minutes. The sequencesrun using 20° C. and even 37° C. labeling steps have distinct patternsof multiple priming events. The sequence run using a 47° C. labelingstep is essentially free of double priming. Similar results wereobtained in the absence of Mn²⁺. Thus, primers which may be toonon-specific to use under low stringency conditions may be specific andusable under more stringent conditions.

Example 7 Increasing Termination Reaction Temperature

There may also be some reason to prefer running the terminationreactions at elevated temperatures as well. Polymerizations will be morerapid at the elevated temperatures, and some template secondarystructures may be eliminated at temperatures above 50° C. Sequences ofsingle- and double-stranded template DNAs can be obtained withtermination reaction temperatures up to 70° C. with T7 DNA polymerase.These termination reaction temperatures are made reliable by theaddition of glycerol to the reaction mixture. (See FIG. 7)

Referring to FIG. 7, sequencing reactions were run using a 96-well plateformat kit (described in example 8) so that the termination reactionscontained 20% glycerol. Labeling reactions were done at 20° C.,termination reactions were pre-warmed at the indicated temperatures.Template was either M13mp18(M13) or alkali-denatured BlueScript(Plasmid). Note that there is little difference in performance usingtermination reaction temperatures from 20° C. to 60° C. Highertemperatures should improve performance when templates with strongsecondary structures are used.

There are yet other situations where it is more convenient to tolerateglycerol in DNA sequencing samples. For instance, one way to sequencelarge linear DNAs is to cut them with a restriction enzyme and gene 6exonuclease. Both of these enzymes are typically supplied in 50%glycerol, so the glycerol content of the template and subsequentsequencing reactions is increased.

In these and perhaps many other situations, it is important to havesequencing gels available which can tolerate loading of glycerol alongwith the DNA sample. This can be readily and inexpensively achieved bychanging the buffer in the sequencing gel to one which does not reactwith glycerol. This opens up many new possibilities for makingsequencing reactions both more convenient and more reliable.

Kits

SEQUENASE and TAQuence kits presently require dilution of enzyme 8-foldprior to use. The present invention allows production of a kit in whichthe reagents are re-formulated to contain enzyme at 1/8th the normalconcentration in a 50%-glycerol solution so that the kit enzyme can beused directly without dilution. Such a kit will save the user time, andpossibly allow the user more actual sequences per kit and more flexibleuse of the enzyme in situations where only 1 or 2 sequences are beingperformed on a given day. Since the sequencing reaction products willcontain large quantities of glycerol, a glycerol-tolerant gelformulation of this invention, or the extra work of removing glycerolfrom the products will be required.

A new kit can also be produced, perhaps in a 96-well plate format, whichconsists of pre-dispensed reagents for sequencing. The enzyme can bedispensed at its final working concentration in 50% glycerol buffer, inwhich it is stable even for a 15-20 minute incubation at 37° C. Otherreaction components whose volumes are critical can also be dispensedusing 30-50% glycerol which prevents the component from freezing underthe plate storage condition (-20° C.) and prevents loss by evaporation(or lyophilization) which occurs when covered plates are frozen.Components whose volume are not critical can simply be dispensed in theplate in convenient amounts. A possible configuration is as follows:

    ______________________________________                                               1   2     3     4   5   6   7   8   9   10  11  12                     ______________________________________                                        Enzyme   ◯                                                                       ◯                                                                       ◯                                                                     ◯                                                                     ◯                                                                     ◯                                                                     ◯                                                                     ◯                                                                     ◯                                                                     ◯                                                                     ◯                                         ◯                                                                 Labeling Mix ◯ ◯ .lar                                 gecircle. ◯ ◯ .largec                                 ircle. ◯ ◯ .largecirc                                 le. ◯ ◯ ◯                                 .                                                                             Stop Solution ◯ ◯ .la                                 rgecircle. ◯ ◯ .large                                 circle. ◯ ◯ .largecir                                 cle. ◯ ◯ .largecircle                                 .                                                                             Buffer ◯ ◯ .largecirc                                 le. ◯ ◯ ◯                                  ◯ ◯ ◯ .l                                 argecircle. ◯ ◯                                       ddGTP Mix ◯ ◯ .largec                                 ircle. ◯ ◯ .largecirc                                 le. ◯ ◯ ◯                                  ◯ ◯ ◯                                    ddATP Mix ◯ ◯ .largec                                 ircle. ◯ ◯ .largecirc                                 le. ◯ ◯ ◯                                  ◯ ◯ ◯                                    ddTTP Mix ◯ ◯ .largec                                 ircle. ◯ ◯ .largecirc                                 le. ◯ ◯ ◯                                  ◯ ◯ ◯                                    ddCTP Mix ◯ ◯ .largec                                 ircle. ◯ ◯ .largecirc                                 le. ◯ ◯ ◯                                  ◯ ◯ ◯       ______________________________________                                    

The template DNA and primer are added to the buffer well and the entireplate incubated at 37° C. for 10 minutes to anneal. Then the labellednucleotide, labeling mix and enzyme are added to the buffer well and thelabeling step incubated for about 5 minutes at room temperature. Thetemperature of the plate is increased to 37° C. and one quarter of themixture then transferred to each of the ddNTP mixes in the lower wellsfor the termination step (at least 5 minutes). Finally, stop solution istransferred from its well to the termination reactions. Since the timingof the steps is not critical, up to 12 templates can be sequenced atonce. When fewer templates are to be sequenced, an appropriate area ofthe plate can simply be cut with scissors and used.

For automated pipetting devices, it is critical that the final totalvolume of the labeling reaction be known, so evaporation or condensationof the buffer, enzyme and labeling mix during cold storage must beminimized. This can be accomplished by the addition of 30-50% glycerolor ethylene glycol which prevents freezing and evaporation orlyophilization. Since the sequencing reaction products will containlarge quantities of glycerol, a glycerol-tolerant gel formulation or theextra work of removing glycerol from the products will be required.

Example 8 SEQUENASE DNA Sequencing Plate Kit

This plate-format kit is designed to run 12 sequences at a time. All ofthe reagents required except template DNA, primer, and radioactivenucleotide are contained on the plate. The plates include reagents forsequencing with 7-deaza-dGTP to eliminate compression artifacts.

High concentrations of glycerol are used to preserve the enzyme activityand the volumes of the labeling mixture and buffer. These highconcentrations of glycerol would normally interfere with the sequencinggel, but the use of a new glycerol-tolerant sequencing gel bufferconveniently and inexpensively eliminates this problem.

The plate is prepared in 12 columns of 8 different reagents (see above).Each column is sufficient for one DNA sequence. The compositions andvolumes of each of the reagents is given below. The enzyme is given alight blue color and the labeling mixture a light red color with dyes.These help to visually verify the actions of the program. The StopSolution is dark blue since it contains electrophoresis tracking dyes.

The protocol is as follows:

1. Prepare 12 template DNA samples (13 μl). These must containapproximately 1 μg of M13 or 3-5 μg of denatured plasmid DNA in 13 μl ofTE buffer.

2. Add 1 μl primer to each DNA sample. The primer should be 0.5-5pmol/μl concentration. The control "-40" M13 primer included is 0.5pmol/μl.

3. Add 0.5 μl (5 μCi) of ³⁵ S dATP (˜1000 Ci/mmol) to each DNA sample.

4. Remove a plate from the freezer, keeping it upright. Place it on thebench at room temperature and carefully remove the tape-like plasticcover. This cover may be saved for re-sealing the plate later.

5. Transfer the entire volume (14.5 μl) of each template/primer toindividual buffer wells on the plate as illustrated. ##STR1## 6.Annealing: Incubate the entire plate at 37° C. for 10 minutes(optimally, the enzyme wells can be kept cooler by extending them offthe surface of the heating block).

7. Cool on an ice block 1 minute.

8. Labeling Reaction: Transfer 5 μl of labeling mix (Red, row B) to thetemplate well. Similarly transfer enzyme (Blue, row A) to the reactionwell. Mix well. Place plate at room temperature 3-5 minutes.

9. Pre-warm entire plate on 37° C. block 1 minute.

10. Termination Reactions: Transfer 5 μl of labeling reaction to each ofthe termination wells (rows E-H), mix well. Incubate 5 minutes or longerat 37° C.

11. Stop: Transfer 5 μl of Stop Solution (dark Blue, row C) to eachtermination reaction, mix well.

12. Cover the plate with a tape sealer and store in the freezer ifdesired.

13. When the glycerol-tolerant sequencing gel is ready to load, uncoverthe plate and place it on top of a heating block set to 70°-80° C. toheat the samples. Heat 3-4 minutes, avoiding excessive evaporation. Loadas soon as possible after heating.

14. Load the samples as sets of 4 adjacent lanes as illustrated.##STR2##

The labeling and termination steps can be run at room temperature withno apparent difficulty. Annealing at room temperature (10 minutes) canbe achieved using at least 2:1 primer:template mole ratio.

Plates can be readily cut with scissors for running fewer than 12reactions.

Labeling reactions can be run as warm as 45° C. for up to 5 minutes.This may be useful for increasing the primer specificity in cases wherethe primer may anneal at more than one place. Termination reactions canbe run at temperatures as high as 60° C. if the labelling reaction ismaintained at room temperature. To do this, the termination wells arecut off the plate, and preincubated at the high temperature for a fewseconds before addition of the labeling reaction mix.

    ______________________________________                                        Reagent Compositions:                                                         ______________________________________                                        1.    Enzyme      5 μl 0.65 Units/μl Ver. 2.0 in 50%                                              glycerol buffer (Bromcresol                                                   Green, 0.02%)                                       2.    Lab. Mix    5 μl 0.6 μM each 7-deaza-dGTP,                                                  dTTP, dCTP in 40% glycerol                                                    (Phenol Red, 0.02%)                                 3.    Stop Solution                                                                             30 μl                                                                              95% Formamide, 20 mM EDTA,                                                    0.05% Bromophenol Blue, 0.05%                                                 Xylene Cyanol FF                                    4.    Buffer      5 μl 100 mM Tris (7.5), 50 mM                                                      MgCl.sub.2, 125 mM NaCl, 40%                                                  glycerol.                                           5.    ddG         5 μl 50 mM NaCl, 40 μM each dNTP,                                               4 μM ddGTP, 20% Glycerol.                        6.    ddA         5 μl 50 mM NaCl, 40 μM each dNTP,                                               4 μM ddATP, 20% Glycerol.                        7.    ddT         5 μl 50 mM NaCl, 40 μM each dNTP,                                               4 μM ddTTP, 20% Glycerol.                        8.    ddC         5 μl 50 mM NaCl, 40 μM each dNTP,                                               4 μM ddCTP, 20% Glycerol.                        ______________________________________                                    

Other embodiments are within the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 3                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      TTTTCATCGGCATTTTCG18                                                          (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      GCCTACGTTCACAGCTG17                                                           (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      CTATCAGCTGTGAACGTAGGCCTTACCCACAGCTGTGCGTAGCTCATACC50                          __________________________________________________________________________

I claim:
 1. A buffer at a pH between 7.5 and 9.5 consisting essentiallyof Tris and taurine in the presence of a geling agent.
 2. A buffer at apH between 7.5 and 9.5 consisting essentially of Tris, taurine, and EDTAin the presence of a geling agent.